Title

Authors

Date of Completion

Spring 5-1-2016

Thesis Advisor(s)

Joerg Graf

Honors Major

Molecular and Cell Biology

Abstract

The plasmid p1471 was isolated from the ciprofloxacin (Cp) resistant Aeromonas hydrophila, medicinal leech isolate JG1471, which is a strain with a minimum inhibitory concentration (MIC) of > 32 µg/mL Cp. JG1471 carries a 6.8 Kb plasmid which contains Cp resistance gene qnrS2. qnrS2 is a plasmid mediated resistance gene that has been isolated from clinical and environmental sources and has been implicated in increased resistance to the fluoroquinolone (FQ) Cp. To determine the FQ resistance conferred by qnrS2, primers were designed to perform a Gibson Assembly to insert a 1.99 Kb fragment from pKAS46, containing an Escherichia coli origin of replication (R6K), multiple cloning sites (MCS) and a kanamycin (Km) resistance gene into the 6.8 Kb p1471. The R6K origin of replication allows successful plasmid replication in E. coli, while the Km resistance factors allow for selection of both Aeromonas and E. coli strains containing the construct. The presence of the MCS in the construct makes it possible for the plasmid to be used as a shuttle vector in future experiments. The assembled construct, pEL1, was transformed into the E. coli DH5α-λ pir strain. Several assays were then performed to confirm that the plasmid construct was assembled correctly. E. coli showed a marked increase in ciprofloxacin resistance upon addition of pEL1, with a ~8-fold increase in MIC from 0. 032 µg/mL to 0.25 µg/mL. When pEL1 was transformed into a CpS strain of Aeromonas veronii, the MIC showed a 3,000-fold increase, from 0.002 µg/mL to 6 µg/mL, passing the 4 µg/mL clinical benchmark for resistance. This data suggests that the presence of qnrS2 in p1471, a high copy plasmid, may play a substantial role in the CpR of the strain.